Jump to

Abstract

Introduction— Apolipoprotein (apo) A-II is the second most abundant high-density lipoprotein (HDL) apolipoprotein. We assessed the mechanism involved in the altered postprandial triglyceride-rich lipoprotein metabolism of female human apoA-II-transgenic mice (hapoA-II-Tg mice), which results in up to an 11-fold increase in plasma triglyceride concentration. The relationships between apoA-II, HDL composition, and lipoprotein lipase (LPL) activity were also analyzed in a group of normolipidemic women.

Methods and Results— Triglyceride-rich lipoprotein catabolism was decreased in hapoA-II-Tg mice compared to control mice. This suggests that hapoA-II, which was mainly associated with HDL during fasting and postprandially, impairs triglyceride-rich lipoprotein lipolysis. HDL isolated from hapoA-II-Tg mice impaired bovine LPL activity. Two-dimensional gel electrophoresis, mass spectrometry, and immunonephelometry identified a marked deficiency in the HDL content of apoA-I, apoC-III, and apoE in these mice. In normolipidemic women, apoA-II concentration was directly correlated with plasma triglyceride and inversely correlated with the HDL-apoC-II+apoE/apoC-III ratio. HDL-mediated induction of LPL activity was inversely correlated with apoA-II and directly correlated with the HDL-apoC-II+apoE/apoC-III ratio. Purified hapoA-II displaced apoC-II, apoC-III, and apoE from human HDL2. Human HDL3 was, compared to HDL2, enriched in apoA-II but poorer in apoC-II, apoC-III, and apoE.

Conclusion— ApoA-II plays a crucial role in triglyceride catabolism by regulating LPL activity, at least in part, through HDL proteome modulation.

Apolipoprotein (apo) A-II is the second most abundant protein, comprising ≈20% of high-density lipoprotein (HDL) total protein mass; however, its function remains unclear.1–4 Evidence for a role of apoA-II in triglyceride (TG) and free fatty acid (FFA) metabolism was provided by reports that showed a positive correlation between apoA-II synthesis and very-low-density lipoprotein apoB in humans, and that showed the APOA-II locus was linked to a locus controlling plasma levels of apoA-II and FFA in mice and humans.1 More recently, several clinical studies reported an association between the −265T>C allele affecting the D element of the apoA-II promoter, altered plasma apoA-II concentration, and postprandial metabolism of large triglyceride-rich lipoproteins (TRL).5–7 Further evidence for a role of apoA-II in TG metabolism in humans has been provided by studies of controls and cases with coronary artery disease, and in families with familial combined hyperlipidemia.8,9 Both studies showed a direct association between apoA-II and plasma TG levels. However, the mechanism whereby apoA-II affects TG metabolism in humans is largely unknown.

To gain more insight into the role of human apoA-II in TG metabolism, we conducted a detailed study of TRL and HDL in our hapoA-II Tg mice and in a group of normolipidemic women. Our data show that accumulation of hapoA-II increases plasma TG through an impaired HDL induction of lipoprotein lipase (LPL) activity that depends, at least in part, on changes induced in the protein composition of discrete HDL subfractions.

Materials and Methods

The hapoA-II-Tg mice were developed as previously described.14 Twenty-three healthy women were enrolled in the study, which was approved by the Ethics Committee for Clinical Investigation of the Hospital de la Santa Creu i Sant Pau. For further experimental details, see the online data supplement at http://atvb.ahajournals.org.

Results

The hapoA-II-Tg mice used in this study displayed 2.5-fold higher hapoA-II plasma levels than those of normolipidemic humans.1 No differences were found in food consumption among mice of different genotypes (4.1±0.3 in control mice vs 3.9±0.3 grams/day in hapoA-II-Tg). Plasma TG levels in female hapoA-II-Tg mice were maximal between 2 hours and 3 hours after the oral fat gavage (OFG), and at that time were 11-fold higher than those in control mice (Figure 1A). Plasma TG in male hapoA-II-Tg mice were 7.5–fold higher 3 hours after the OFG (5.9±1.0 vs 0.8±0.1 mmol/L in controls). Because females were more prone to hypertriglyceridemia, we conducted a series of experiments in our female hapoA-II-Tg mice. Radiolabeled TG mixed with olive oil was individually gavaged, and the radiolabeled TG was determined in plasma and target tissues at the times indicated (Figure 1B,C). Plasma levels of radiolabeled TG in hapoA-II-Tg mice were significantly higher than those of control mice after 3 hours of OFG administration (Figure 1B). At that time, a very high percentage (>99%) of radioactivity present in the lipoprotein-depleted fraction was mainly attributable to FFA, and these were found to be significantly decreased in hapoA-II-Tg mice (1.5%) compared to control mice (8.9%). The hapoA-II-Tg mice showed decreased radiolabeled TG in parametrial White Adipose Tissue (pWAT) 6 hours after OFG (Figure 1C).

Figure 1. Oral fat gavage (OFG) assays in transgenic (Tg) and knockout (KO) mice. A, TG was measured before and after a single bolus of 150 μL of olive oil in female hapoA-II-Tg and control mice. In separate experiments, radiolabeled TG were, respectively, measured in plasma (B) and in target tissues (C) after a single bolus of 150 μL of radiolabeled olive oil-based emulsion (20 μCi per mouse) at the times indicated. D, TG were also measured before and after a single bolus of 150 μL of olive oil in female apoA-I KO and apoA-II KO mice. Results are expressed as mean±SEM of individual animals (n=5 mice at each time point). *P<0.05 compared with the control.

Because hypertriglyceridemia is common in patients with inherited HDL deficiency, and because hapoA-II is known to displace mouse apoA-I from the HDL surface,1 we determined postprandial plasma TG profiles after an OFG in female apoA-I KO and apoA-II KO mice (Figure 1D). Although potential changes in postprandial TG could be observed and were consistent with previous studies,12,15 the area under the curve of TG after the OFG in apoA-I KO and apoA-II KO mice did not significantly differ from that of control mice (Figure 1D). This may be because of the limited number of animals studied. However, overall, these results suggested that the increased postprandial TG in hapoA-II-Tg mice was not attributable to their decreased HDL.

Postprandial plasma hapoA-II concentration in the hapoA-II-Tg mice used in the present study (82.6±5.3 mg/dL) did not differ significantly from that found in fasting plasma (75.0±4.2 mg/dL). Fast protein liquid chromatography (FPLC) analyses of postprandial plasma revealed a dramatic increase in TG and cholesterol amount and size of the TRL particles of hapoA-II-Tg mice compared with those of control mice (Figure 2A, B, respectively). Decreased HDL cholesterol in hapoA-II-Tg mice was not more pronounced postprandially than in fasting conditions (Figure 2B). The hapoA-II was mainly found in the HDL fraction (77.8±5.51 mg/dL), but small amounts could be detected in chylomicrons (3.1±0.5 mg/dL; Figure 2C, D). Sixty-eight percent of plasma TG in hapoA-II-Tg mice floated as chylomicrons (Figure 2D). The TG-to-protein ratio confirmed that postprandial plasma transgenic TRL were significantly larger than those of control TRL (online Figure I).

Effect of hapoA-II Expression on TRL Plasma Turnover in Mice

In vivo analyses were performed to evaluate the causes of postprandial hypertriglyceridemia in hapoA-II-Tg mice. Data indicated that TG intestinal and liver secretion in hapoA-II-Tg mice did not significantly differ from those of control mice (Figure 3A, B). In vivo catabolic studies with autologous [3H]-TG-TRL showed that the fractional catabolic rate of hapoA-II-Tg-mice [3H]-TG-TRL was significantly reduced in plasma of hapoA-II-Tg mice compared to that of control mice under postprandial conditions (Figure 3C, D). In contrast, [3H]-TG-TRL fractional catabolic rate did not significantly differ between genotypes under fasting conditions. Changes in TG clearance in hapoA-II-Tg mice under postprandial conditions may reflect the dilution of [3H]-TG-TRL in an expanded TRL pool. For this reason, we also performed cross-infusion experiments consisting of the injection of labeled TRL from transgenic mice into control mice, and labeled TRL from control into hapoA-II -Tg mice (see the 2 last bars of Figure 3D). Data indicated that control [3H]-TG-TRL clearance from plasma of hapoA-II-Tg mice was significantly delayed compared to that in control mice, whereas hapoA-II-Tg [3H]-TG-TRL were cleared from plasma of control mice as rapidly as the autologous control [3H]-TG-TRL (Figure 3D). This could indicate that the plasma content, rather than TRL origin, was the main determinant of the changes in TRL catabolism. Although postheparin postprandial or fasting plasma LPL activity toward an artificial triolein-based substrate in hapoA-II-Tg mice did not differ from control mice (Figure 3E), coincubation with HDL of control mice increased bovine LPL activity 60% more than coincubation with apoA-II-Tg mice HDL (Figure 3F). In addition, we conducted experiments to test the ability of HDL from hapoA-II-Tg mice and control mice to modulate in vitro bovine LPL activity toward TRL (data not shown). Coincubation of TRL with HDL from control mice and hapoA-II-Tg mice at ratios similar to those found in vivo in each group of mice (Figures 1 and supplemental Figure I) resulted in 4-fold more enzymatic activity in the former condition compared with the latter. To rule out the possibility that the redistribution of hapoA-II in chylomicrons could affect LPL activity, we incubated control chylomicrons with free hapoA-II at 37°C for 3 hours. The rate of FFA formation in apoA-II-enriched chylomicrons, at a hapoA-II concentration of 3 mg/dL (which represents ≈4% of the total concentration of proteins in chylomicrons), was similar to that of control chylomicrons (13.8±0.9 vs 11.9±0.7 nmol of FFA/mL per minute).

Figure 3. Effect of hapoA-II on in vivo intestinal and liver TG secretion, autologous and heterologous TRL catabolism, and LPL activity. A, In vivo intestinal TG secretion in overnight fasted hapoA-II-Tg and control mice given an OFG of [3H]-TG. B, In vivo liver TG secretion of mice after intravenous injection of 10 μCi of [3H]-oleic acid. C, Plasma clearance of autologous [3H]-TRL-TG, isolated during fasting or 3 hours after an OFG and injected intravenously. D, Plasma [3H]-TG clearance of (C) displayed as fractional catabolic rate (first 4 bars). The last 2 bars correspond to heterologous infusions (performed 3 hours after OFG): [3H]-TG-labeled TRL of transgenic mice were injected into control mice and control [3H]-TG-labeled TRL were injected into hapoA-II transgenic mice. E, LPL activity in postheparin plasma in mice. Each value represents the mean±SEM of data from 5 mice. F, Bovine LPL activity against a tri[9,10(n)-3H] oleate-based emulsion incubated with mouse HDL samples at 37°C for 60 minutes. The SEM at each point are indicated unless they fall within the bar. With the exception of (E), results are expressed as mean±SEM of at least 3 independent experiments with pools of 3 to 4 animals. *P<0.05 vs HDL from control mice.

Effect of hapoA-II on Plasma and HDL-Associated Apolipoproteins in Mice

HDL protein composition was studied by 2-dimensional gel electrophoresis. This analysis showed the existence of 10 significant, differentially expressed spots (Figure 4A) and 2 hapoA-II isoforms in apoA-II-Tg mice HDL. Identification of these spots was performed by Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Figure 4A, B). Two spots contained a mixture of paraoxonase 1 and an apoA-I isoform; thus, the respective contributions to their decreased abundance could not be determined. To confirm these changes, we measured several available murine apolipoproteins by immunonephelometry in plasma and in HDL obtained by ultracentrifugation (Figure 4C, D). This method confirmed that the amount of apoC-III was almost negligible in hapoA-II-Tg mice HDL, whereas plasma apoC-III levels were 3-fold higher in postprandial conditions, thereby indicating a redistribution of this apolipoprotein to TRL (Figure 4C, D). Again, apoA-I was decreased in plasma and in HDL in fasting and postprandial conditions (Figure 4C, D).

Human Studies

In a group of healthy normolipidemic women (n=23), hapoA-II concentration was found to be directly correlated with plasma TG (r=0.72; P<0.001; Figure 5A). HDL of these women was incubated in the presence of a triolein-based emulsion and bovine LPL. ApoA-II was found to be inversely correlated with HDL-mediated induction of LPL activity (r=−0.45; P<0.04; Figure 5B). HDL modulation of LPL activity was also directly correlated with the HDL-apoC-II+apoE/apoC-III, HDL-apoC-II/apoC-III, and apoA-V/apoA-I ratios (see supplemental Table II for detailed correlation coefficients); however, only the first parameter significantly correlated with apoA-II (r=−0.41; P<0.05; Figure 5C).

Effect of hapoA-II on Human HDL Apolipoprotein Content

Coincubation of human HDL2 with purified hapoA-II clearly decreased the intensity of the immunoreactive bands, both in denaturing and nondenaturing conditions, corresponding to human apoC-II, apoC-III, and apoE when analyzed by Western blot (Figure 6A). This incubation also generated a novel peak that contained mainly apoA-II and apoA-I (data not shown). ApoA-II was preferentially located in HDL3, and this was concomitant with a strongly decreased content of apoC-II, apoC-III, and apoE in HDL3 compared to that of HDL2 (Figure 6B).

Figure 6. Effect of hapoA-II on human HDL apolipoprotein composition. A, FPLC profiles of native human HDL2 (apoA-I concentration of 30 mg/dL) before and after incubation with 60 mg/dL of purified hapoA-II. The results of specific immunoblots of fraction 67 are shown both in denaturing and nondenaturing conditions. B, Relative apolipoprotein concentration of HDL2 and HDL3 isolated by ultracentrifugation of 7 pools of normolipidemic plasma. Indicated apolipoproteins were determined by immunonephelometry.

Discussion

Evidence favoring a role for apoA-II in TG metabolism stems from experiments in mice1–4,10,11 and, also, human studies.1,5–9 However, the precise mechanism by which hapoA-II influences TG metabolism remains unclear. In the current study, we show that overexpression of human apoA-II in transgenic mice increased postprandial plasma TG concentration. A similar degree of postprandial hypertriglyceridemia was also found in the double hapoA-IIxCETP-Tg mice having a hapoA-II plasma concentration of 42 mg/dL,16 thereby indicating that the effect of hapoA-II on TG metabolism also occurs at more physiologically significant levels. Because liver and intestinal TG secretion was comparable between hapoA-II-Tg mice and control mice, we focused our study on postprandial TRL catabolism. Our data clearly show that the hypertriglyceridemia of hapoA-II-Tg mice was primarily caused by a decrease in TRL clearance. An important question is whether postprandial plasma TG accumulation resulted from the association of hapoA-II with TRL. In a previous report using independently generated hapoA-II-Tg mice,13 severe postprandial hypertriglyceridemia was associated with a marked increase in plasma hapoA-II concentration in TRL particles, which were found to be poor LPL substrates.10 In contrast, plasma hapoA-II of our hapoA-II-Tg mice was mainly found associated with HDL during the postprandial state, and we found no nearly complete depletion of the HDL fraction nor a fed-to-fasted switch in hapoA-II concentration. It is possible that the different hapoA-II gene constructions used to generate these 2 hapoA-II-Tg mice1,13 could explain their differences in hapoA-II plasma concentration regulation in these 2 different transgenic models. Increased catabolism and decreased production of HDL were the main mechanisms leading to the partial HDL deficiency in our hapoA-II-Tg mice.17 These changes seemed to be more severe in the other transgenic model,18 leading to severe hapoA-II accumulation in TRL. Taken together, our data indicate that the amount of hapoA-II present in TRL in our hapoA-II-Tg mice did not impair its catabolism.

ApoA-II variability was also correlated with the concentration of TG in healthy normolipidemic women with hapoA-II levels ranging from 22.5 to 45.4 mg/dL. In agreement with data from our hapoA-II-Tg, hapoA-II concentration was found to be directly correlated with plasma TG 3 hours after the fat-loading test (r=0.59; P<0.05) in 13 subjects who were given a fatty meal with 1 gram of fat per kilogram of body weight, whereas that with HDL cholesterol was only close to significance (r=−0.51; P=0.073). Importantly, plasma apoA-II levels were unaffected by the fat-loading test (34.8±1.4 vs 35.6±1.5 mg/dL under fasting conditions), and TRL contained negligible amounts of apoA-II. This finding strongly suggests that the marked increase in hapoA-II in TRL that leads to LPL inhibition is not a physiological mechanism in humans. Nevertheless, a direct LPL inhibition by apoA-II may indeed occur in pathological situations, such as hypertriglyceridemia associated with Tangier disease and type V hyperlipidemia in humans,1 in cases of extreme HDL deficiency in apoA-II-Tg mice,10,19 and when apoA-II accumulates in TRL, as in mouse apoA-II-Tg mice.11 This and other major differences between human and mouse apoA-II, such as the divergent effect on HDL levels, had been attributed to species-specific differences in amino acid sequence, dimer formation, and affinity for lipoprotein surfaces.1 The reported inhibition of LPL by apoA-II may also be extendable to other lipases, such as hepatic lipase and endothelial lipase.12,20

Data obtained in our hapoA-II-Tg mice suggested that the presence of hapoA-II in HDL might influence HDL apolipoprotein composition or apolipoprotein transfer to TRL, thereafter altering TRL postprandial catabolism. To test this hypothesis in detail, LPL assays were performed using mouse HDL from each genotype as the activator apolipoprotein source. Our results clearly indicated that the ability of hapoA-II HDL to modulate LPL activity was lower than that induced by control HDL. We also found that apoA-II levels showed an inverse correlation with the capacity of HDL to induce LPL activity in healthy normolipidemic volunteers in absence of plasma as a source of LPL-activating apolipoproteins, and this parameter was also strongly correlated with the HDL-apoC-II+apoE/apoC-III ratio. ApoC-II is well-known to be an effective activator of LPL activity, whereas apoC-III acts as an inhibitor.21 ApoE may also exert enhancing effects on the action of LPL, although apoE is mainly a ligand of different cellular lipoproteins.22 It is also well-known that at least part of apoC-II, apoC-III, and apoE is acquired by TRL from the HDL reservoir during the postprandial state; thus, the alteration in HDL composition during fasting may strongly affect TRL metabolism and plasma TG levels.23–25 Therefore, it is conceivable that an altered concentration and, therefore, transfer of these HDL apolipoproteins impaired TG catabolism in individuals with high hapoA-II levels. The hapoA-II-Tg mice provided a useful model to gain more insight into this question. Proteomic and immunonephelometric analyses of the HDL of hapoA-II-Tg mice did demonstrate a decreased content in apoA-I and paraoxonase 1, a deficiency previously reported to be caused by displacement of the HDL surface by a high hapoA-II content,26,27 and also demonstrated a deficiency in the HDL content of apoC-III and apoE. Thus, it is possible that hapoA-II-Tg mice HDL is a poor acceptor for apoC-III, thereby causing redistribution to TRL during the postprandial state, which impairs LPL activity. Alternatively, apoC-III accumulation in postprandial TRL of hapoA-II-Tg mice could be the consequence, rather than the cause, of the hypertriglyceridemia. ApoC-II, the main cofactor required for LPL activity, was not identified by proteomic analysis and could not be measured by immunonephelometry. Previous reports showed that HDL3 presented a higher apoA-II-to-apoA-I ratio than HDL2,28,29 whereas apoC-II, apoC-III, and apoE were found to be mainly associated with HDL2.28,30,31 We therefore analyzed whether human HDL subfractions enriched with apoA-II were poorer in apoC-II, apoC-III, and apoE. This was the case, as demonstrated by quantifying these apolipoproteins in pools of HDL2 and HDL3. In an attempt to ascertain whether hapoA-II could affect apoC-II content and whether there was a common mechanism responsible for the rest of the changes found in HDL apolipoproteins, we incubated purified hapoA-II with human HDL2 and found that the increased association of hapoA-II with HDL2 caused a decrease in the content of apoC-II, apoC-III, and apoE. The displacement of these apolipoproteins, especially apoC-II, from HDL and the consequent altered transfer to TRL may thus explain, at least in part, the association found between hapoA-II with TG and with the capacity of HDL to activate LPL, both in mice and humans.1,26 These results also demonstrate that hapoA-II is a major regulator of the apoC-II, apoC-III, and apoE distribution in discrete HDL particle subfractions, as previously shown with paraoxonase 1.26 This is likely to be attributable to the higher affinity of hapoA-II for the HDL surface compared with these apolipoproteins.1,26 Therefore, although TRL apolipoprotein composition is recognized as modulator TG metabolism, our results indicate that HDL composition could play an active role in this process. Further, based on our observations, we speculate that the relative decrease in HDL2 found in hypertriglyceridemia29 could play an active role in its pathophysiology by decreasing LPL activity.

In conclusion, our study shows that hapoA-II excess in HDL may contribute to postprandial hypertriglyceridemia by inhibiting TRL lipolysis mediated by LPL attributable, at least in part, to apoC-II, apoC-III, and apoE displacement from the HDL surface. Because the results of this displacement are consistent with the differential distribution of apoA-II and apoC-II, apoC-III, and apoE in HDL2 and HDL3, it is suggested that hapoA-II has a role in determining the proteome of discrete HDL particle subfractions.

Acknowledgments

Sources of Funding

This work was funded in part by FIS grants 06/0551, 07/1067, and 08/1147, and by AGAUR 2009-SGR-1205. CIBER de Diabetes y Enfermedades Metabólicas Asociadas, CIBERDEM, is a project of the Instituto de Salud Carlos III.

Patsch JR, Prasad S, Gotto AM Jr, Patsch W. High density lipoprotein2. Relationship of the plasma levels of this lipoprotein species to its composition, to the magnitude of postprandial lipemia, and to the activities of lipoprotein lipase and hepatic lipase. J Clin Invest. 1987; 80: 341–347.